Development of a quantitative assay for mitochondrial fusion and characterization of a lipid signaling pathway on the mitochondrial surface

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Abstract

Mitochondria continuously undergo fusion and fission, the relative rates of which define their morphology. Mitochondrial fusion is currently assayed by fusing together cells expressing GFP or RFP in their mitochondria and then scoring the frequency of cells with yellow mitochondria (representing fused green and red mitochondria). However, this assay is labor intensive and only semi-quantitative. I developed a reporter system consisting of split fragments of Renilla luciferase and YFP fused to mitochondrial matrix-targeting sequences and to leucine zippers to trigger dimerization. The assay enables fusion to be quantitated both visually for individual cells and on a population level using chemiluminescence, laying the foundation for high throughput small molecule and RNAi screens for modulators of mitochondrial fusion. I used the assay to examine cytoskeletal roles in fusion progression. The mammalian Phospholipase D superfamily member MitoPLD localizes to mitochondria and facilitates fusion by generating the signaling lipid phosphatidic acid (PA). Independently, the Drosophila homolog of MitoPLD, Zucchini (Zuc), was proposed to be a cytoplasmic nuclease and shown to be required for piRNA generation, a critical step in oogenesis. I show here that Zuc localizes to mitochondria. Conversely, mice lacking MitoPLD exhibit the germ cell degeneration observed in mice lacking other piRNA components. These findings identify the first mitochondrial protein required for piRNA generation. MitoPLD generates PA on the mitochondrial surface. The PA then recruits another signaling enzyme, the PA phosphatase Lipin 1, to convert the PA to diacylglycerol and promote fission, suggesting a new mechanism for mitochondrial morphology homeostasis. MitoPLD<super>-/-</super> fibroblasts exhibit shortened mitochondria and spermatocytes lack intermitochondrial cement (nuage), a structure that is implicated in piRNA generation. I propose that mitochondrial-surface PA generated by MitoPLD/Zuc recruits nuage components critical for piRNA production.